A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis

Abstract

The Wnt/β-catenin signaling pathway is a highly conserved pathway in organism evolution and regulates many biological processes. Aberrant activation of the Wnt/β-catenin signaling pathway is closely related to tumorigenesis. In order to identify potent small molecules to treat the over-activated Wnt signaling-mediated cancer, such as colon cancer, we established a mammalian cell line-based reporter gene screening system. The screen revealed a diterpenoid derivative, 15-oxospiramilactone (NC043) that inhibits Wnt3a or LiCl-stimulated Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and Caco-2. Treatment of SW480 cells with NC043 led to decreases in the mRNA and/or protein expression of Wnt target genes Axin2, Cyclin D1 and Survivin , as well as decreases in the protein levels of Cdc25c and Cdc2. NC043 did not affect the cytosol-nuclear distribution and protein level of soluble β-catenin, but decreased β-catenin/TCF4 association in SW480 cells. Moreover, NC043 inhibited anchorage-independent growth and xenograft tumorigenesis of SW480 cells. Collectively these results demonstrate that NC043 is a novel small molecule that inhibits canonical Wnt signaling downstream of β-catenin stability and may be a potential compound for treating colorectal cancer.

Figure 1

NC043 inhibits the Wnt/β-catenin signaling reporter Top-flash. (A) NC043 inhibits Top-flash activity in a dose-dependent manner in HEK293T cells. DMSO or NC043 with indicated dosage was added to cells 17 h after transfection with the Top-flash plasmid. One hour later, the cells were treated with control or Wnt3a conditioned medium (CM) or 20 mM of LiCl containing the same dosage of NC043 for additional 6 h. Luciferase activity was then measured. (B) NC043 inhibits Top-flash activity in a dose-dependent manner in colon carcinoma cells, SW480 and Caco-2. Cells were treated with NC043 for 24 h. Data represent the mean±S.D. from three independent experiments. (C) The chemical structure of NC043 and its derivatives. (D) The effect of NC043 derivatives on Top-flash activity in SW480 cells. Transfected SW480 cells were treated with NC043 and its derivatives and incubated for 24 h. Data represent the mean±S.D. from one experiment. Experiments were repeated three times. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, significant relative to vehicle control. NS, no statistics significance.

Figure 2

NC043 selectively inhibits Wnt signaling and its target genes expression. (A) NC043 specifically inhibits Top-flash activity. DMSO or NC043 was added to the cells 17 hrs after transfection. One hour after the incubation of NC043 (7.5 μM), Wnt3a CM for Top-flash, Ionomycin (1 μg/ml) for NF-AT luc and Forskolin (10 μM) for CRE luc (all of them containing the same dosage of NC043) were added for additional 6 h. (B, C) NC043 inhibits mRNA and protein expressions of Axin2 , Survivin and Cyclin D1. SW480 cells was incubated with DMSO (shorten for D) or NC043 with indicated dosage for 3 h. Samples were then prepared for qPCR (B) or western blot (C). Data represent the mean±S.D. from one experiment. Experiments were repeated three times. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, significant relative to vehicle control. NS, no statistics significance.

Figure 3

NC043 does not affect β-catenin stability and distribution but does impair β-catenin/TCF4 association. (A) NC043 has no effect on β-catenin protein expression and distribution in HEK293T cells. Cells were incubated with NC043 or DMSO. After 1 h cells were treated with control or Wnt3a CM containing the same dosage of NC043 for additional 3 h. The cells were then fractioned as described in Materials and Methods, and the samples were analyzed by western blot. β-tubulin was used as a cytosolic marker and loading control. SP1 was used as a nuclear marker and loading control. (B, C) NC043 but not its derivatives impair β-catenin/TCF4 association. HEK293T cells (B) were incubated with NC043 and its derivatives. One hour later, cells were treated with Wnt3a conditioned medium (CM) containing the same dosage of small molecules for additional 3 h. SW480 cells (C) were incubated with NC043 or DMSO. Nuclear fractions were immunoprecipitated by the β-catenin antibody and analyzed by western blot. For quantitative western blot analysis, the integrated intensity of each signal was measured. The TCF4/β-catenin signal ratios were shown. (D) NC043 does not affect β-catenin and LEF1 interaction in vitro. Biosensor Binding Studies were performed as described in Material and Methods. The equilibrium response data for 6His-β-catenin was fitted to 1:1 binding site model to obtain the K_D of ∼1.84 μM. NC043 was then added with 6His-β-catenin. The equilibrium response data for NC043 was the same compared to the data without NC043. Data are representative of one experiment. Experiments were repeated at least three times. (E) NC043 does not affect β-catenin/E-cadherin association. SW480 cells were incubated with NC043 for 1 h. Whole cell lysates were then immunoprecipiataed by an E-cadherin antibody and analyzed by western blot. For quantitative western blot analysis, the integrated intensity of each signal was measured. The β-catenin/E-cadherin signal ratios were shown. (F) NC043 inhibits ΔN-β-catenin but not ΔN-LEF-VP16 induced Top-flash activity. DMSO or NC043 and its derivatives with indicated dosage was added to HEK293T cells for 6 h after transfection with the Top-flash, LacZ, ΔN-β-catenin and ΔN-LEF-VP16 plasmids. Luciferase activity was then measured. Data were calculated as the fold activation induced by ΔN-β-catenin and ΔN-LEF-VP16 compared with control plasmid (LacZ) and presented the mean±S.D. from three independent experiments. (G) NC043 depresses the recruitment of β-catenin but not TCF to the Cyclin D1 promotor. SW480 cells were incubated with 30 μM of NC043 for 2 h and a ChIP assay was performed using the β-catenin and TCF4 antibodies. The product of ChIP was then analyzed by qPCR. Data represent the mean±S.E. from at least three independent experiments. ^***P < 0.001, significant relative to vehicle control. NS, no statistics significance.

Figure 4

NC043 causes G2/M arrest and inhibition of colon cancer cells growth. (A) The effect of NC043 and its derivatives on cell growth of colon carcinoma cells (SW480 and Caco-2) or normal colonic epithelial cells (CCD-841-CoN). Cells were incubated with chemicals and then analyzed by MTT assay. IC₅₀ of 72 h is shown. Data represent the mean±S.E. from three independent experiments. (B) NC043 arrests SW480 cells at G2/M phase of the cell cycle. SW480 cells were incubated with NC043 for 36 h, and were stained with PI and analyzed by FACS. Data represent the mean±S.D. from two independent experiments. (C) NC043 decreases Cdc25c and Cdc2 protein expression in a dose-dependent manner.

Figure 5

NC043 inhibits SW480 cell tumorigenesis in vitro and in vivo. (A, B) NC043 but not its derivatives inhibit anchorage-independent growth of SW480 cells. SW480 cells were seeded in soft agar with NC043 (A) and its derivatives (B) for 18 days and stained with crystal violet. Data represent the mean±S.E. from three independent experiments. (C-F) NC043 inhibits SW480 cell tumorigenesis in a xenograft model. Tumor bearing mice were treated intraperitoneally with either vehicle or NC043 (45 and 90 μg/kg) daily for 17 days. Body weight (C), tumor size (E) and tumor weight (F) were measured as described in Materials and Methods. The external appearance of tumors is shown (D). Data represent the mean±S.E., ^*P < 0.05, ^**P < 0.01, significant relative to vehicle control.